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Absolute Protein Quantification Using AQUA-Calibrated 2D-PAGE

  • Sandra Maaß
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1841)

Abstract

Absolute protein quantification for the analysis of proteome dynamics is more and more required by the scientific community. Therefore a number of methods have recently been reported that aim at determining concentrations of single proteins in complex samples, all of them having their advantages and limitations. However, for all of these methods an accurate and protein unspecific determination of the total protein amount in a given sample is urgently needed. Here a ninhydrin-based assay established to reach this goal is described. Moreover, an optimized protocol for protein digestion is an inevitable prerequisite for all mass spectrometry-based approaches aiming at absolute protein quantification. In this chapter, various aspects are described which have to be considered during validation of a suitable digestion method and a detailed protocol is presented that can be applied to the digestion of soluble proteins originated from microbes.

In order to provide an absolute protein quantification workflow applicable for small scale and large scale approaches, a step-by-step guide is provided for the so-called AQUA-strategy (AQUA = absolute quantification), including selection of suited standard peptides, the development of optimized MS methods and the determination of absolute protein concentration using stable isotope dilution and selected reaction monitoring (SID-SRM). Subsequently, a workflow is introduced that combines targeted mass spectrometry and two-dimensional polyacrylamide gel electrophoresis for the large-scale determination of absolute protein amounts.

Key words

Proteomics Absolute protein quantification Protein Digestion AQUA Stable isotope dilution Selected reaction monitoring 2D-PAGE 

Notes

Acknowledgments

This work was supported by the German Research Foundation Grant SFB/TR34.

References

  1. 1.
    Bennett EJ, Rush J, Gygi SP, Harper JW (2010) Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 143(6):951–965CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kuepfer L, Peter M, Sauer U, Stelling J (2007) Ensemble modeling for analysis of cell signaling dynamics. Nat Biotechnol 25(9):1001–1006CrossRefPubMedGoogle Scholar
  3. 3.
    Holzmann J, Pichler P, Madalinski M, Kurzbauer R, Mechtler K (2009) Stoichiometry determination of the MP1-p14 complex using a novel and cost-efficient method to produce an equimolar mixture of standard peptides. Anal Chem 81(24):10254–10261CrossRefPubMedGoogle Scholar
  4. 4.
    Muntel J, Fromion V, Goelzer A, Maaβ S, Mäder U, Büttner K, Hecker M, Becher D (2014) Comprehensive absolute quantification of the cytosolic proteome of Bacillus subtilis by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE). Mol Cell Proteomics 13(4):1008–1019CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Nanavati D, Gucek M, Milne JLS, Subramaniam S, Markey SP (2008) Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope-labeled concatenated peptide standards. Mol Cell Proteomics 7(2):442–447CrossRefPubMedGoogle Scholar
  6. 6.
    Schmidt C, Lenz C, Grote M, Lührmann R, Urlaub H (2010) Determination of protein stoichiometry within protein complexes using absolute quantification and multiple reaction monitoring. Anal Chem 82(7):2784–2796CrossRefPubMedGoogle Scholar
  7. 7.
    Wepf A, Glatter T, Schmidt A, Aebersold R, Gstaiger M (2009) Quantitative interaction proteomics using mass spectrometry. Nat Methods 6(3):203–205CrossRefPubMedGoogle Scholar
  8. 8.
    Lu P, Vogel C, Wang R, Yao X, Marcotte EM (2007) Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nat Biotechnol 25(1):117–124CrossRefPubMedGoogle Scholar
  9. 9.
    Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M (2011) Global quantification of mammalian gene expression control. Nature 473(7347):337–342CrossRefPubMedGoogle Scholar
  10. 10.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  11. 11.
    Compton SJ, Jones CG (1985) Mechanism of dye response and interference in the Bradford protein assay. Anal Biochem 151(2):369–374CrossRefPubMedGoogle Scholar
  12. 12.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275Google Scholar
  13. 13.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85CrossRefPubMedGoogle Scholar
  14. 14.
    Starcher B (2001) A ninhydrin-based assay to quantitate the total protein content of tissue samples. Anal Biochem 292(1):125–129CrossRefPubMedGoogle Scholar
  15. 15.
    Maass S, Sievers S, Zühlke D, Kuzinski J, Sappa PK, Muntel J, Hessling B, Bernhardt J, Sietmann R, Völker U, Hecker M, Becher D (2011) Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal Chem 83(7):2677–2684CrossRefPubMedGoogle Scholar
  16. 16.
    Piehowski PD, Petyuk VA, Orton DJ, Xie F, Moore RJ, Ramirez-Restrepo M, Engel A, Lieberman AP, Albin RL, Camp DG, Smith RD, Myers AJ (2013) Sources of technical variability in quantitative LC-MS proteomics: human brain tissue sample analysis. J Proteome Res 12(5):2128–2137CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Cañas B, Piñeiro C, Calvo E, López-Ferrer D, Gallardo JM (2007) Trends in sample preparation for classical and second generation proteomics. J Chromatogr A 1153(1-2):235–258CrossRefPubMedGoogle Scholar
  18. 18.
    Switzar L, Giera M, Niessen WMA (2013) Protein digestion: an overview of the available techniques and recent developments. J Proteome Res 12(3):1067–1077CrossRefPubMedGoogle Scholar
  19. 19.
    Norrgran J, Williams TL, Woolfitt AR, Solano MI, Pirkle JL, Barr JR (2009) Optimization of digestion parameters for protein quantification. Anal Biochem 393(1):48–55CrossRefPubMedGoogle Scholar
  20. 20.
    Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100(12):6940CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kirkpatrick DS, Gerber SA, Gygi SP (2005) The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 35(3):265–273CrossRefPubMedGoogle Scholar
  22. 22.
    Anderson L, Hunter CL (2006) Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics 5(4):573–588CrossRefPubMedGoogle Scholar
  23. 23.
    Keshishian H, Addona T, Burgess M, Kuhn E, Carr SA (2007) Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics 6(12):2212–2229CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kuhn E, Wu J, Karl J, Liao H, Zolg W, Guild B (2004) Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics 4(4):1175–1186CrossRefPubMedGoogle Scholar
  25. 25.
    Picotti P, Bodenmiller B, Mueller LN, Domon B, Aebersold R (2009) Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138(4):795–806CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lange V, Malmstrom JA, Didion J, King NL, Johansson BP, Schafer J, Rameseder J, Wong CH, Deutsch EW, Brusniak MY (2008) Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring. Mol Cell Proteomics 7(8):1489CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Desiere F, Deutsch EW, Nesvizhskii AI, Mallick P, King NL, Eng JK, Aderem A, Boyle R, Brunner E, Donohoe S, Fausto N, Hafen E, Hood L, Katze MG, Kennedy KA, Kregenow F, Lee H, Lin B, Martin D, Ranish JA, Rawlings DJ, Samelson LE, Shiio Y, Watts JD, Wollscheid B, Wright ME, Yan W, Yang L, Yi EC, Zhang H, Aebersold R (2005) Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry. Genome Biol 6(1):R9CrossRefPubMedGoogle Scholar
  28. 28.
    Deutsch EW, Lam H, Aebersold R (2008) PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows. EMBO Rep 9(5):429–434CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Prince JT, Carlson MW, Wang R, Lu P, Marcotte EM (2004) The need for a public proteomics repository. Nat Biotechnol 22(4):471–472CrossRefPubMedGoogle Scholar
  30. 30.
    Mallick P, Schirle M, Chen SS, Flory MR, Lee H, Martin D, Ranish J, Raught B, Schmitt R, Werner T, Kuster B, Aebersold R (2007) Computational prediction of proteotypic peptides for quantitative proteomics. Nat Biotechnol 25(1):125–131CrossRefPubMedGoogle Scholar
  31. 31.
    Tang H, Arnold RJ, Alves P, Xun Z, Clemmer DE, Novotny MV, Reilly JP, Radivojac P (2006) A computational approach toward label-free protein quantification using predicted peptide detectability. Bioinform Oxf Engl 22(14):e481–e488CrossRefGoogle Scholar
  32. 32.
    Mayya V, Rezual K, Wu L, Fong MB, Han DK (2006) Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry: determination of inhibitory phosphorylation status of cyclin-dependent kinases. Mol Cell Proteomics 5(6):1146–1157CrossRefPubMedGoogle Scholar
  33. 33.
    Brun V, Dupuis A, Adrait A, Marcellin M, Thomas D, Court M, Vandenesch F, Garin J (2007) Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol Cell Proteomics 6(12):2139–2149CrossRefPubMedGoogle Scholar
  34. 34.
    Hanke S, Besir H, Oesterhelt D, Mann M (2008) Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level. J Proteome Res 7(3):1118–1130CrossRefPubMedGoogle Scholar
  35. 35.
    Singh S, Springer M, Steen J, Kirschner MW, Steen H (2009) FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. J Proteome Res 8(5):2201–2210CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Zeiler M, Straube WL, Lundberg E, Uhlen M, Mann M (2012) A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines. Mol Cell Proteomics 11(3):O111.009613CrossRefPubMedGoogle Scholar
  37. 37.
    Ong S-E, Foster LJ, Mann M (2003) Mass spectrometric-based approaches in quantitative proteomics. Methods San Diego CA 29(2):124–130CrossRefGoogle Scholar
  38. 38.
    Büttner K, Bernhardt J, Scharf C, Schmid R, Mäder U, Eymann C, Antelmann H, Völker A, Völker U, Hecker M (2001) A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis. Electrophoresis 22(14):2908–2935CrossRefPubMedGoogle Scholar
  39. 39.
    Berth M, Moser FM, Kolbe M, Bernhardt J (2007) The state of the art in the analysis of two-dimensional gel electrophoresis images. Appl Microbiol Biotechnol 76(6):1223–1243CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Kuntumalla S, Braisted JC, Huang S-T, Parmar PP, Clark DJ, Alami H, Zhang Q, Donohue-Rolfe A, Tzipori S, Fleischmann RD, Peterson SN, Pieper R (2009) Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Leptospira interrogans proteome. Proteome Sci 7:22CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Bantscheff M, Lemeer S, Savitski MM, Kuster B (2012) Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal Bioanal Chem 404(4):939–965CrossRefPubMedGoogle Scholar
  42. 42.
    Maaß S, Becher D (2016) Methods and applications of absolute protein quantification in microbial systems. J Proteome 136:222–233CrossRefGoogle Scholar
  43. 43.
    Bandow JE, Baker JD, Berth M, Painter C, Sepulveda OJ, Clark KA, Kilty I, VanBogelen RA (2008) Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies—COPD biomarker discovery study. Proteomics 8(15):3030–3041CrossRefPubMedGoogle Scholar
  44. 44.
    Kraut A, Marcellin M, Adrait A, Kuhn L, Louwagie M, Kieffer-Jaquinod S, Lebert D, Masselon CD, Dupuis A, Bruley C, Jaquinod M, Garin J, Gallagher-Gambarelli M (2009) Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. J Proteome Res 8(7):3778–3785CrossRefPubMedGoogle Scholar
  45. 45.
    van Midwoud PM, Rieux L, Bischoff R, Verpoorte E, Niederländer HAG (2007) Improvement of recovery and repeatability in liquid chromatography-mass spectrometry analysis of peptides. J Proteome Res 6(2):781–791CrossRefPubMedGoogle Scholar
  46. 46.
    Stahl-Zeng J, Lange V, Ossola R, Eckhardt K, Krek W, Aebersold R, Domon B (2007) High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites. Mol Cell Proteomics 6(10):1809–1817CrossRefPubMedGoogle Scholar
  47. 47.
    Loziuk PL, Sederoff RR, Chiang VL, Muddiman DC (2014) Establishing ion ratio thresholds based on absolute peak area for absolute protein quantification using protein cleavage isotope dilution mass spectrometry. Analyst 139(21):5439–5450CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Maaß S, Wachlin G, Bernhardt J, Eymann C, Fromion V, Riedel K, Becher D, Hecker M (2014) Highly precise quantification of protein molecules per cell during stress and starvation responses in Bacillus subtilis. Mol Cell Proteomics 13(9):2260–2276CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Microbial Proteomics, Institute for MicrobiologyUniversity GreifswaldGreifswaldGermany

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